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Numerous studies could prove that children with reading disorders show RAN deficits and that RAN is especially strongly connected to the automatization of word recognition. In contrast intervention studies so far have provided little convincing evidence for the trainability of RAN or even a transfer effect on reading competence. The aim of this study is therefore to evaluate the efficacy of an adaptive, software-based RAN-training in a group of 57 children aged 9.3 years (SD 1.10 years) with a RAN-Deficit and/or difficulties with word-reading-speed and to determine if there is a transfer effect to reading speed of words and pseudowords as well as to reading comprehension on the word-, sentence and text level. The software-based training of RAN letters, numbers and colors is an intervention over 18 training days. Children were encouraged to name visually presented items in a serial format as fast as a red rectangle jumping from item to item. The speed of the red rectangle was successively increased with each successful training session. Univariate ANOVAs with repeated measures showed that children had a significantly higher naming speed for letters and numbers both immediately after completion of the intervention and in the follow-up measures. A transfer of the training effect to reading speed for words, reading comprehension on the word- and sentence-level, but not to reading speed for pseudowords and text comprehension, could be demonstrated. Due to the missing of a control-group comparison, it remains to be proven in subsequent research whether this effect is a maturation-related change or a training effect.

Abstract Static gene expression programs have been extensively characterized in stem cells and mature human cells. However, the dynamics of RNA isoform changes upon cell-state-transitions during cell differentiation, the determinants and functional consequences have largely remained unclear. Here, we established an improved model for human neurogenesis in vitro that is amenable for systems-wide analyses of gene expression. Our multi-omics analysis reveals that the pronounced alterations in cell morphology correlate strongly with widespread changes in RNA isoform expression. Our approach identifies thousands of new RNA isoforms that are expressed at distinct differentiation stages. RNA isoforms mainly arise from exon skipping and the alternative usage of transcription start and polyadenylation sites during human neurogenesis. The transcript isoform changes can remodel the identity and functions of protein isoforms. Finally, our study identifies a set of RNA binding proteins as a potential determinant of differentiation stage-specific global isoform changes. This work supports the view of regulated isoform changes that underlie state-transitions during neurogenesis.

Abstract Autophagy-related protein 18 (Atg18) participates in the elongation of early autophagosomal structures in concert with Atg2 and Atg9 complexes. How Atg18 contributes to the structural coordination of Atg2 and Atg9 at the isolation membrane remains to be understood. Here, we determined the cryo-EM structures of Atg18 organized in helical tubes, Atg18 oligomers in solution as well as on lipid membrane scaffolds. The helical assembly is composed of Atg18 tetramers forming a lozenge cylindrical lattice with remarkable structural similarity to the COPII outer coat. When reconstituted with lipid membranes, using subtomogram averaging we determined tilted Atg18 dimer structures bridging two juxtaposed lipid membranes spaced apart by 80 Å. Moreover, lipid reconstitution experiments further delineate the contributions of Atg18’s FRRG motif and the amphipathic helical extension in membrane interaction. The observed structural plasticity of Atg18’s oligomeric organization and membrane binding properties provide a molecular framework for the positioning of downstream components of the autophagy machinery.

BackgroundPersistent radiological lung abnormalities are evident in many survivors of acute coronavirus disease 2019 (COVID-19). Consolidation and ground glass opacities are interpreted to indicate subacute inflammation whereas reticulation is thought to reflect fibrosis. We sought to identify differences at molecular and cellular level, in the local immunopathology of post-COVID inflammation and fibrosis.MethodsWe compared single-cell transcriptomic profiles and T cell receptor (TCR) repertoires of bronchoalveolar cells obtained from convalescent individuals with each radiological pattern, targeting lung segments affected by the predominant abnormality.ResultsCD4 central memory T cells and CD8 effector memory T cells were significantly more abundant in those with inflammatory radiology. Clustering of similar TCRs from multiple donors was a striking feature of both phenotypes, consistent with tissue localised antigen-specific immune responses. There was no enrichment for known SARS-CoV-2-reactive TCRs, raising the possibility of T cell-mediated immunopathology driven by failure in immune self-tolerance.ConclusionsPost-COVID radiological inflammation and fibrosis show evidence of shared antigen-specific T cell responses, suggesting a role for therapies targeting T cells in limiting post-COVID lung damage.

Abstract Gene transcription by RNA polymerase II (Pol II) is under control of promoters and distal regulatory elements known as enhancers. Enhancers are themselves transcribed by Pol II correlating with their activity. How enhancer transcription is regulated and coordinated with transcription at target genes has remained unclear. Here, we developed a high-sensitive native elongating transcript sequencing approach, called HiS-NET-seq, to provide an extended high-resolution view on transcription, especially at lowly transcribed regions such as enhancers. HiS-NET-seq uncovers new transcribed enhancers in human cells. A multi-omics analysis shows that genome-wide enhancer transcription depends on the BET family protein BRD4. Specifically, BRD4 co-localizes to enhancer and promoter-proximal gene regions, and is required for elongation activation at enhancers and their genes. BRD4 keeps a set of enhancers and genes in proximity through long-range contacts. From these studies BRD4 emerges as a general regulator of enhancer transcription that may link transcription at enhancers and genes.

Abstract Many proteins involved in eukaryotic phosphate homeostasis are regulated by SPX domains. In yeast, the vacuolar transporter chaperone (VTC) complex contains two such domains, but mechanistic details of its regulation are not well understood. Here, we show at the atomic level how inositol pyrophosphates interact with SPX domains of subunits Vtc2 and Vtc3 to control the activity of the VTC complex. Vtc2 inhibits the catalytically active VTC subunit Vtc4 by homotypic SPX–SPX interactions via the conserved helix α1 and the previously undescribed helix α7. Binding of inositol pyrophosphates to Vtc2 abrogates this interaction, thus activating the VTC complex. Accordingly, VTC activation is also achieved by site-specific point mutations that disrupt the SPX–SPX interface. Structural data suggest that ligand binding induces reorientation of helix α1 and exposes the modifiable helix α7, which might facilitate its post-translational modification in vivo. The variable composition of these regions within the SPX domain family might contribute to the diversified SPX functions in eukaryotic phosphate homeostasis.

Eukaryotic cells control inorganic phosphate to balance its role as essential macronutrient with its negative bioenergetic impact on reactions liberating phosphate. Phosphate homeostasis depends on the conserved INPHORS signaling pathway that utilizes inositol pyrophosphates and SPX receptor domains. Since cells synthesize various inositol pyrophosphates and SPX domains bind them promiscuously, it is unclear whether a specific inositol pyrophosphate regulates SPX domains in vivo, or whether multiple inositol pyrophosphates act as a pool. In contrast to previous models, which postulated that phosphate starvation is signaled by increased production of the inositol pyrophosphate 1-IP7, we now show that the levels of all detectable inositol pyrophosphates of yeast, 1-IP7, 5-IP7, and 1,5-IP8, strongly decline upon phosphate starvation. Among these, specifically the decline of 1,5-IP8 triggers the transcriptional phosphate starvation response, the PHO pathway. 1,5-IP8 inactivates the cyclin-dependent kinase inhibitor Pho81 through its SPX domain. This stimulates the cyclin-dependent kinase Pho85-Pho80 to phosphorylate the transcription factor Pho4 and repress the PHO pathway. Combining our results with observations from other systems, we propose a unified model where 1,5-IP8 signals cytosolic phosphate abundance to SPX proteins in fungi, plants, and mammals. Its absence triggers starvation responses.

Abstract Exome sequencing has introduced a paradigm shift for the identification of germline variations responsible for Mendelian diseases. However, non‐coding regions, which make up 98% of the genome, cannot be captured. The lack of functional annotation for intronic and intergenic variants makes RNA‐seq a powerful companion diagnostic. Here, we illustrate this point by identifying six patients with a recessive Osteogenesis Imperfecta (OI) and neonatal progeria syndrome. By integrating homozygosity mapping and RNA‐seq, we delineated a deep intronic TAPT1 mutation (c.1237‐52 G>A) that segregated with the disease. Using SI‐NET‐seq, we document that TAPT1's nascent transcription was not affected in patients' fibroblasts, indicating instead that this variant leads to an alteration of pre‐mRNA processing. Predicted to serve as an alternative splicing branchpoint, this mutation enhances TAPT1 exon 12 skipping, creating a protein‐null allele. Additionally, our study reveals dysregulation of pathways involved in collagen and extracellular matrix biology in disease‐relevant cells. Overall, our work highlights the power of transcriptomic approaches in deciphering the repercussions of non‐coding variants, as well as in illuminating the molecular mechanisms of human diseases.

ABSTRACT Cells stabilize intracellular inorganic phosphate (Pi) to compromise between large biosynthetic needs and detrimental bioenergetic effects of Pi. Pi homeostasis in eukaryotes uses Syg1/Pho81/Xpr1 (SPX) domains, which are receptors for inositol pyrophosphates. We explored how polymerization and storage of Pi in acidocalcisome-like vacuoles supports Saccharomyces cerevisiae metabolism and how these cells recognize Pi scarcity. Whereas Pi starvation affects numerous metabolic pathways, beginning Pi scarcity affects few metabolites. These include inositol pyrophosphates and ATP, a low-affinity substrate for inositol pyrophosphate-synthesizing kinases. Declining ATP and inositol pyrophosphates may thus be indicators of impending Pi limitation. Actual Pi starvation triggers accumulation of the purine synthesis intermediate 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR), which activates Pi-dependent transcription factors. Cells lacking inorganic polyphosphate show Pi starvation features already under Pi-replete conditions, suggesting that vacuolar polyphosphate supplies Pi for metabolism even when Pi is abundant. However, polyphosphate deficiency also generates unique metabolic changes that are not observed in starving wild-type cells. Polyphosphate in acidocalcisome-like vacuoles may hence be more than a global phosphate reserve and channel Pi to preferred cellular processes. IMPORTANCE Cells must strike a delicate balance between the high demand of inorganic phosphate (Pi) for synthesizing nucleic acids and phospholipids and its detrimental bioenergetic effects by reducing the free energy of nucleotide hydrolysis. The latter may stall metabolism. Therefore, microorganisms manage the import and export of phosphate, its conversion into osmotically inactive inorganic polyphosphates, and their storage in dedicated organelles (acidocalcisomes). Here, we provide novel insights into metabolic changes that yeast cells may use to signal declining phosphate availability in the cytosol and differentiate it from actual phosphate starvation. We also analyze the role of acidocalcisome-like organelles in phosphate homeostasis. This study uncovers an unexpected role of the polyphosphate pool in these organelles under phosphate-rich conditions, indicating that its metabolic roles go beyond that of a phosphate reserve for surviving starvation.

Summary: T cell responses precede antibody and may provide early control of infection. We analyzed the clonal basis of this rapid response following SARS-COV-2 infection. We applied T cell receptor (TCR) sequencing to define the trajectories of individual T cell clones immediately. In SARS-COV-2 PCR+ individuals, a wave of TCRs strongly but transiently expand, frequently peaking the same week as the first positive PCR test. These expanding TCR CDR3s were enriched for sequences functionally annotated as SARS-COV-2 specific. Epitopes recognized by the expanding TCRs were highly conserved between SARS-COV-2 strains but not with circulating human coronaviruses. Many expanding CDR3s were present at high frequency in pre-pandemic repertoires. Early response TCRs specific for lymphocytic choriomeningitis virus epitopes were also found at high frequency in the preinfection naive repertoire. High-frequency naive precursors may allow the T cell response to respond rapidly during the crucial early phases of acute viral infection.

The “anti-branching rule”, introduced in 1950, excludes branched polyphosphates from biological relevance due to their supposedly rapid hydrolysis. Here, the authors synthesize monodisperse branched polyphosphates and demonstrate their unexpected stability in water, as well as provide evidence for their competence in phosphorylation.

Hosts diverge widely in how, and how well, they defend themselves against infection and immunopathology. Why are hosts so heterogeneous? Both epidemiology and life history are commonly hypothesized to influence host immune strategy, but the relationship between immune strategy and each factor has commonly been investigated in isolation. Here, we show that interactions between life history and epidemiology are crucial for determining optimal immune specificity and sensitivity. We propose a demographically-structured population dynamics model, in which we explore sensitivity and specificity of immune responses when epidemiological risks vary with age. We find that variation in life history traits associated with both reproduction and longevity alters optimal immune strategies-but the magnitude and sometimes even direction of these effects depends on how epidemiological risks vary across life. An especially compelling example that explains previously-puzzling empirical observations is that depending on whether infection risk declines or rises at reproductive maturity, later reproductive maturity can select for either greater or lower immune specificity, potentially illustrating why studies of lifespan and immune variation across taxa have been inconclusive. Thus, the sign of selection on the life history-immune specificity relationship can be reversed in different epidemiological contexts. Drawing on published life history data from a variety of chordate taxa, we generate testable predictions for this facet of the optimal immune strategy. Our results shed light on the causes of the heterogeneity found in immune defenses both within and among species and the ultimate variability of the relationship between life history and immune specificity.

Summary: Lysosome functions mainly rely on their ability to either degrade substrates or release them into the extracellular space. Lysosomal storage disorders (LSDs) are commonly characterized by a chronic lysosomal accumulation of different substrates, thereby causing lysosomal dysfunctions and secretion defects. However, the early effects of substrate accumulation on lysosomal homeostasis have not been analyzed so far. Here, we describe how the acute accumulation of a single substrate determines a rapid centripetal redistribution of the lysosomes, triggering their expansion and reducing their secretion, by limiting the motility of these organelles toward the plasma membrane. Moreover, we provide evidence that such defects could be explained by a trapping mechanism exerted by the extensive contacts between the enlarged lysosomes and the highly intertwined membrane structures of the endoplasmic reticulum which might represent a crucial biological cue ultimately leading to the clinically relevant secondary defects observed in the LSD experimental models and patients.

Abstract Short rotation plantations are often considered as holding vast potentials for future global bioenergy supply. In contrast to raising biomass harvests in forests, purpose‐grown biomass does not interfere with forest carbon (C) stocks. Provided that agricultural land can be diverted from food and feed production without impairing food security, energy plantations on current agricultural land appear as a beneficial option in terms of renewable, climate‐friendly energy supply. However, instead of supporting energy plantations, land could also be devoted to natural succession. It then acts as a long‐term C sink which also results in C benefits. We here compare the sink strength of natural succession on arable land with the C saving effects of bioenergy from plantations. Using geographically explicit data on global cropland distribution among climate and ecological zones, regionally specific C accumulation rates are calculated with IPCC default methods and values. C savings from bioenergy are given for a range of displacement factors (DFs), acknowledging the varying efficiency of bioenergy routes and technologies in fossil fuel displacement. A uniform spatial pattern is assumed for succession and bioenergy plantations, and the considered timeframes range from 20 to 100 years. For many parameter settings—in particular, longer timeframes and high DFs—bioenergy yields higher cumulative C savings than natural succession. Still, if woody biomass displaces liquid transport fuels or natural gas‐based electricity generation, natural succession is competitive or even superior for timeframes of 20–50 years. This finding has strong implications with climate and environmental policies: Freeing land for natural succession is a worthwhile low‐cost natural climate solution that has many co‐benefits for biodiversity and other ecosystem services. A considerable risk, however, is C stock losses (i.e., emissions) due to disturbances or land conversion at a later time.

The adaptive immune system responds to pathogens by selecting clones of cells with specific receptors. While clonal selection in response to particular antigens has been studied in detail, it is unknown how a lifetime of exposures to many antigens collectively shape the immune repertoire. Here, using mathematical modeling and statistical analyses of T cell receptor sequencing data, we develop a quantitative theory of human T cell dynamics compatible with the statistical laws of repertoire organization. We find that clonal expansions during a perinatal time window leave a long-lasting imprint on the human T cell repertoire, which is only slowly reshaped by fluctuating clonal selection during adult life. Our work provides a mechanism for how early clonal dynamics imprint the hierarchy of T cell clone sizes with implications for pathogen defense and autoimmunity.

Cells face major changes in demand for and supply of inorganic phosphate (Pi). Pi is often a limiting nutrient in the environment, particularly for plants and microorganisms. At the same time, the need for phosphate varies, establishing conflicts of goals. Cells experience strong peaks of Pi demand, e.g., during the S-phase, when DNA, a highly abundant and phosphate-rich compound, is duplicated. While cells must satisfy these Pi demands, they must safeguard themselves against an excess of Pi in the cytosol. This is necessary because Pi is a product of all nucleotide-hydrolyzing reactions. An accumulation of Pi shifts the equilibria of these reactions and reduces the free energy that they can provide to drive endergonic metabolic reactions. Thus, while Pi starvation may simply retard growth and division, an elevated cytosolic Pi concentration is potentially dangerous for cells because it might stall metabolism. Accordingly, the consequences of perturbed cellular Pi homeostasis are severe. In eukaryotes, they range from lethality in microorganisms such as yeast (Sethuraman et al., 2001; Hürlimann, 2009), severe growth retardation and dwarfism in plants (Puga et al., 2014; Liu et al., 2015; Wild et al., 2016) to neurodegeneration or renal Fanconi syndrome in humans (Legati et al., 2015; Ansermet et al., 2017). Intracellular Pi homeostasis is thus not only a fundamental topic of cell biology but also of growing interest for medicine and agriculture.

Protozoan parasites contain negatively charged polymers of a few up to several hundreds of phosphate residues. In other organisms, these polyphosphate (polyP) chains serve as an energy source and phosphate reservoir, and have been implicated in adaptation to stress and virulence of pathogenic organisms. In this study, we confirmed first that the polyP polymerase vacuolar transporter chaperone 4 (VTC4) is responsible for polyP synthesis in Leishmania parasites. During Leishmania in vitro culture, polyP is accumulated in logarithmic growth phase and subsequently consumed once stationary phase is reached. However, polyP is not essential since VTC4-deficient (vtc4–) Leishmania proliferated normally in culture and differentiated into infective metacyclic parasites and into intracellular and axenic amastigotes. In in vivo mouse infections, L. major VTC4 knockout showed a delay in lesion formation but ultimately gave rise to strong pathology, although we were unable to restore virulence by complementation to confirm this phenotype. Knockdown of VTC4 did not alter the course of L. guyanensis infections in mice, suggesting that polyP was not required for infection, or that very low levels of it suffice for lesion development. At higher temperatures, Leishmania promastigotes highly consumed polyP, and both knockdown or deletion of VTC4 diminished parasite survival. Thus, although polyP was not essential in the life cycle of the parasite, our data suggests a role for polyP in increasing parasite survival at higher temperatures, a situation faced by the parasite when transmitted to humans.

Metal bioavailability is frequently limited by sequestering agents which makes them inaccessible to cells. Here the authors show that cells can increase Mg2+ uptake via fluid phase endocytosis and accumulate this metal in their vacuole loaded with polyphosphate, and later can be exported to the cytosol.

There is ample evidence that an unabated growth in material consumption is likely to pass the earth system's source and sink capacities. In the face of limited resources, distributional questions increasingly gain importance. Material flow accounting is a methodological tool to trace biophysical patterns of disproportionate resource consumption across countries and the debt towards the environment, other parts of the world, and towards future generations through the excessive consumption of natural resources. At the core of this article, we address different developments of material use for individual countries and world regions from 1950 to 2010. During this phase, fossil fuel-based industrialization triggered an unprecedented growth in material consumption, mainly in the wealthy world regions of Europe, Australia, North America, and partly in the countries of the former Soviet Union, while low resource consumption persists in other regions. We thus calculated cumulative resource use from 1950 to 2010 to show the extent of this wealth built up upon countries' own resources, or through imports from other countries or world regions. We use the degree of net-import dependency of individual countries as a proxy for the ecological debt, and relate it to the domestic resource extraction in a country. Our observations show that there was a highly uneven distribution of resource extraction and use in the 60 years analyzed, which has important implications for future global resource policies. Keywords: Ecological debt, material flow accounting, international trade, global resource use Résumé Il

Public concern and complaints regarding ambient air in zones of dense traffic pertains to two compounds of nitrogen, nitrogen dioxide (NO2) and ammonia (NH3); both are toxic and strongly irritant, such that legal limitations are under discussion. This paper contributes to measuring methods as already in part proposed by GRPE subgroup WLTP-DTP (Worldwide Light Duty Test Procedures – Diesel Test Procedures) for NO2. Despite legally lowered NOx emission levels, lumping both, NO2 and NO, levels of NO2 have risen in cities and agglomerations as a result of both, deployed catalytic exhaust after-treatment devices and low sulphur Diesel fuels. In present tests two different combinations of NO2 measuring methods as proposed by WLTP were checked on Diesel cars for practicability in handling and accuracy. These integral, indirect methods (NO2 = NOx – NO) have been found as useful tools for estimate of NO2 and with use of appropriate analyzers a satisfactory accuracy was attained. Furthermore, attention was brought to ammonia (NH3) emitted by gasoline engines with three way catalysts (TWC) which ought not to be ignored while on the other hand SCR systems for Diesel engines are strictly regulated. Emission levels of more recent TWC turned out to be mostly below 20 ppm NH3. Vehicle of older technology exhibited significantly higher levels, about 10 times more. As chemical reactions depend on pressure and temperature (= i.e. flow condition in CVS-tunnel) as well as concentrations, doubts need to be considered on accuracy of results based on chemical reactive substances. Nevertheless, clear tendencies regarding changes of concentrations of NO2 and NH3 along the path-way could not be observed.

Safeguarding existing forests is an important ecological concern but constrains the expansion of farmland to feed the growing world population. Here the authors analyse the option space for future changes in agriculture and diets compatible with a no-deforestation goal.

Global bioenergy potentials have been the subject of extensive research and continued controversy. Due to vast uncertainties regarding future yields, diets and other influencing parameters, estimates of future agricultural biomass potentials vary widely. Most scenarios compatible with ambitious climate targets foresee a large expansion of bioenergy, mainly from energy crops that needs to be kept consistent with projections of agriculture and food production. Using the global biomass balance model BioBaM, we here present an assessment of agricultural bioenergy potentials compatible with the Food and Agriculture Organization’s (2018) ‘Alternative pathways to 2050’ projections. Mobilizing biomass at larger scales may be associated with systemic feedbacks causing greenhouse gas (GHG) emissions, e.g. crop residue removal resulting in loss of soil carbon stocks and increased emissions from fertilization. To assess these effects, we derive ‘GHG cost supply-curves’, i.e. integrated representations of biomass potentials and their systemic GHG costs. Livestock manure is most favourable in terms of GHG costs, as anaerobic digestion yields reductions of GHG emissions from manure management. Global potentials from intensive livestock systems are about 5 EJ/yr. Crop residues can provide up to 20 EJ/yr at moderate GHG costs. For energy crops, we find that the medium range of literature estimates (∼40 to 90 EJ/yr) is only compatible with FAO yield and human diet projections if energy plantations expand into grazing areas (∼4–5 million km ^2 ) and grazing land is intensified globally. Direct carbon stock changes associated with perennial energy crops are beneficial for climate mitigation, yet there are—sometimes considerable—‘opportunity GHG costs’ if one accounts the foregone opportunity of afforestation. Our results indicate that the large potentials of energy crops foreseen in many energy scenarios are not freely and unconditionally available. Disregarding systemic effects in agriculture can result in misjudgement of GHG saving potentials and flawed climate mitigation strategies.

As long as economic growth is a major political goal, decoupling growth from resource use and emissions is a prerequisite for a sustainable net-zero emissions future. However, empirical evidence for absolute decoupling, i.e. decreasing resource use and emissions at the required scale despite continued economic growth, is scarce and scattered across different research streams. In this two-part systematic review, we assess how and to what extent decoupling has been observed and what can be learnt for addressing the sustainability and climate crisis. Based on a transparent approach, we systematically identify and screen more than 11 500 scientific papers, eventually analyzing full texts of 835 empirical studies on the relationship between economic growth (GDP), resource use (materials and energy) and greenhouse gas emissions. Part I of the review examines how decoupling has been investigated across three research streams: energy, materials and energy, and emissions. Part II synthesizes the empirical evidence and policy implications (Haberl et al 2020 Environ. Res. Lett. 15 065003). In part I, we examine the topical, temporal and geographical scopes, methods of analysis, institutional networks and prevalent conceptual angles. We find that in this rapidly growing literature, the vast majority of studies—decomposition, ‘causality’ and Environmental Kuznets Curve analysis—approach the topic from a statistical-econometric point of view, while hardly acknowledging thermodynamic principles on the role of energy and materials for socio-economic activities. A potentially fundamental incompatibility between economic growth and systemic societal changes to address the climate crisis is rarely considered. We conclude that the existing wealth of empirical evidence merits braver conceptual advances than we have seen thus far. Future work should focus on comprehensive multi-indicator long-term analyses, conceptually grounded on the fundamental biophysical basis of socio-economic activities, incorporating the role of global supply chains as well as the wider societal role and preconditions of economic growth.

Strategies toward ambitious climate targets usually rely on the concept of ‘decoupling’; that is, they aim at promoting economic growth while reducing the use of natural resources and GHG emissions. GDP growth coinciding with absolute reductions in emissions or resource use is denoted as ‘absolute decoupling’, as opposed to ‘relative decoupling’, where resource use or emissions increase less so than does GDP. Based on the bibliometric mapping in part I (Wiedenhofer et al , 2020 Environ. Res. Lett. 15 https://doi.org/10.1088/1748-9326/ab8429 ), we synthesize the evidence emerging from the selected 835 peer-reviewed articles. We evaluate empirical studies of decoupling related to final/useful energy, exergy, use of material resources, as well as CO _2 and total GHG emissions. We find that relative decoupling is frequent for material use as well as GHG and CO _2 emissions but not for useful exergy, a quality-based measure of energy use. Primary energy can be decoupled from GDP largely to the extent to which the conversion of primary energy to useful exergy is improved. Examples of absolute long-term decoupling are rare, but recently some industrialized countries have decoupled GDP from both production- and, weaklier, consumption-based CO _2 emissions. We analyze policies or strategies in the decoupling literature by classifying them into three groups: (1) Green growth, if sufficient reductions of resource use or emissions were deemed possible without altering the growth trajectory. (2) Degrowth, if reductions of resource use or emissions were given priority over GDP growth. (3) Others, e.g. if the role of energy for GDP growth was analyzed without reference to climate change mitigation. We conclude that large rapid absolute reductions of resource use and GHG emissions cannot be achieved through observed decoupling rates, hence decoupling needs to be complemented by sufficiency-oriented strategies and strict enforcement of absolute reduction targets. More research is needed on interdependencies between wellbeing, resources and emissions.

Abstract To obtain rates of mRNA synthesis and decay in yeast, we established dynamic transcriptome analysis (DTA). DTA combines non‐perturbing metabolic RNA labeling with dynamic kinetic modeling. DTA reveals that most mRNA synthesis rates are around several transcripts per cell and cell cycle, and most mRNA half‐lives range around a median of 11 min. DTA can monitor the cellular response to osmotic stress with higher sensitivity and temporal resolution than standard transcriptomics. In contrast to monotonically increasing total mRNA levels, DTA reveals three phases of the stress response. During the initial shock phase, mRNA synthesis and decay rates decrease globally, resulting in mRNA storage. During the subsequent induction phase, both rates increase for a subset of genes, resulting in production and rapid removal of stress‐responsive mRNAs. During the recovery phase, decay rates are largely restored, whereas synthesis rates remain altered, apparently enabling growth at high salt concentration. Stress‐induced changes in mRNA synthesis rates are predicted from gene occupancy with RNA polymerase II. DTA‐derived mRNA synthesis rates identified 16 stress‐specific pairs/triples of cooperative transcription factors, of which seven were known. Thus, DTA realistically monitors the dynamics in mRNA metabolism that underlie gene regulatory systems.

Messenger RNA (mRNA) synthesis and export are tightly linked, but the molecular mechanisms of this coupling are largely unknown. In Saccharomyces cerevisiae, the conserved TREX complex couples transcription to mRNA export and mediates mRNP formation. Here, we show that TREX is recruited to the transcription machinery by direct interaction of its subcomplex THO with the serine 2-serine 5 (S2/S5) diphosphorylated CTD of RNA polymerase II. S2 and/or tyrosine 1 (Y1) phosphorylation of the CTD is required for TREX occupancy in vivo, establishing a second interaction platform necessary for TREX recruitment in addition to RNA. Genome-wide analyses show that the occupancy of THO and the TREX components Sub2 and Yra1 increases from the 5' to the 3' end of the gene in accordance with the CTD S2 phosphorylation pattern. Importantly, in a mutant strain, in which TREX is recruited to genes but does not increase towards the 3' end, the expression of long transcripts is specifically impaired. Thus, we show for the first time that a 5'-3' increase of a protein complex is essential for correct expression of the genome. In summary, we provide insight into how the phospho-code of the CTD directs mRNP formation and export through TREX recruitment.

The equilibrium of membrane fusion and fission influences the volume and copy number of organelles. Fusion of yeast vacuoles has been well characterized but their fission and the mechanisms determining vacuole size and abundance remain poorly understood. We therefore attempted to systematically characterize factors necessary for vacuole fission. Here, we present results of an in vivo screening for deficiencies in vacuolar fragmentation activity of an ordered collection deletion mutants, representing 4881 non-essential genes of the yeast Saccharomyces cerevisiae. The screen identified 133 mutants with strong defects in vacuole fragmentation. These comprise numerous known fragmentation factors, such as the Fab1p complex, Tor1p, Sit4p and the V-ATPase, thus validating the approach. The screen identified many novel factors promoting vacuole fragmentation. Among those are 22 open reading frames of unknown function and three conspicuous clusters of proteins with known function. The clusters concern the ESCRT machinery, adaptins, and lipases, which influence the production of diacylglycerol and phosphatidic acid. A common feature of these factors of known function is their capacity to change membrane curvature, suggesting that they might promote vacuole fragmentation via this property.

SNARE complexes are required for membrane fusion in the endomembrane system. They contain coiled-coil bundles of four helices, three (Q(a), Q(b), and Q(c)) from target (t)-SNAREs and one (R) from the vesicular (v)-SNARE. NSF/Sec18 disrupts these cis-SNARE complexes, allowing reassembly of their subunits into trans-SNARE complexes and subsequent fusion. Studying these reactions in native yeast vacuoles, we found that NSF/Sec18 activates the vacuolar cis-SNARE complex by selectively displacing the vacuolar Q(a) SNARE, leaving behind a Q(bc)R subcomplex. This subcomplex serves as an acceptor for a Q(a) SNARE from the opposite membrane, leading to Q(a)-Q(bc)R trans-complexes. Activity tests of vacuoles with diagnostic distributions of inactivating mutations over the two fusion partners confirm that this distribution accounts for a major share of the fusion activity. The persistence of the Q(bc)R cis-complex and the formation of the Q(a)-Q(bc)R trans-complex are both sensitive to the Rab-GTPase inhibitor, GDI, and to mutations in the vacuolar tether complex, HOPS (HOmotypic fusion and vacuolar Protein Sorting complex). This suggests that the vacuolar Rab-GTPase, Ypt7, and HOPS restrict cis-SNARE disassembly and thereby bias trans-SNARE assembly into a preferred topology.

In eukaryotes, hundreds of mRNAs are localized by specialized transport complexes. For localization, transcripts are recognized by RNA-binding proteins and incorporated into motor-containing messenger ribonucleoprotein particles (mRNPs). To date, the molecular assembly of such mRNPs is not well understood and most details on cargo specificity remain unresolved. We used ASH1-mRNA transport in yeast to provide a first assessment of where and how localizing mRNAs are specifically recognized and incorporated into mRNPs. By using in vitro-interaction and reconstitution assays, we found that none of the implicated mRNA-binding proteins showed highly specific cargo binding. Instead, we identified the cytoplasmic myosin adapter She3p as additional RNA-binding protein. We further found that only the complex of the RNA-binding proteins She2p and She3p achieves synergistic cargo binding, with an at least 60-fold higher affinity for localizing mRNAs when compared to control RNA. Mutational studies identified a C-terminal RNA-binding fragment of She3p to be important for synergistic RNA binding with She2p. The observed cargo specificity of the ternary complex is considerably higher than previously reported for localizing mRNAs. It suggests that RNA binding for mRNP localization generally exhibits higher selectivity than inferred from previous in vitro data. This conclusion is fully consistent with a large body of in vivo evidence from different organisms. Since the ternary yeast complex only assembles in the cytoplasm, specific mRNA recognition might be limited to the very last steps of mRNP assembly. Remarkably, the mRNA itself triggers the assembly of mature, motor-containing complexes. Our reconstitution of a major portion of the mRNA-transport complex offers new and unexpected insights into the molecular assembly of specific, localization-competent mRNPs and provides an important step forward in our mechanistic understanding of mRNA localization in general.

The so-called psychoanalytic setting counts today as a familiar ritual, forming a set of conventions that are weil known, if not from first-hand experience, then at least from popular novels and films. Adopting an historical-anthropological viewpoint, this article unfolds a genealogy of Freud's famous invention. A genealogical approach of this kind has to account for both the social and the material arrangements from which psychoanalysis emerged at the end of the 19th century. lt is argued that Freud's conception of a certain spatial distribution of patient and doctor in the consulting room was not governed by either technical or theoretical necessity, but was rather the result of social and material reconfigurations which can be trac- ed back to the history of experimental hypnotism.